Smooth muscle (SM) cells form the main part of the walls of blood vessels, the airways, the gastrointestinal and reproductive tracts. Pathology of SM contractility plays a key role in hypertension, cerebral and coronary vasospasm, erectile dysfunction, bronchial asthma, and other diseases. SM contractility at a given level of Ca2+ is critically modulated by a complex network of protein-protein interactions, which can enhance the contractile effect acting via a small GTPase RhoA. The design of molecules that would alter these interactions could provide a more specific way of therapeutic targeting of RhoA signaling. It is well understood that RhoA, a ubiquitous molecular switch, is controlled by many different GEFs (guanine nucleotide exchange factors) and GAPs (GTPase activating proteins), which either load RhoA with GTP (GEFs) or downregulate it by catalyzing the hydrolysis of GTP to GDP (GAPs). Which GEFs and which GAPs are active in SM, and how they contribute to the regulation of contractility - is not known. We propose to identify GEFs and GAPs active in SM, and to dissect the mechanisms by which they operate. This is an exciting stage in our ongoing studies of the mechanisms underlying the molecular and structural biology of the RhoA-dependent signaling pathways. Among the GEFs relevant to SM physiology are three RGS RhoGEFs, interacting with the Ga12/13 subunits, for which we have already accumulated a substantial amount of structural information. We present, for the first time, biochemical and functional data implicating p63RhoGEF/GEFT that interacts with Gaq11 linked to specific G-protein-coupled receptors. Promising results of our qRT-PCR experiments identify several GAPs and GEFs new to SM that may down and up regulate RhoA respectively and modulate SM contractility. We also formulate a new hypothesis, supported by preliminary data, which postulates that negative control is exerted on RhoA by cyclic nucleotides (cAMP) acting via the Rap1 GEF, Epac and Rap1 (another GTPase) to activate RhoA specific GAPs including ARAP3 and RA-RhoGAP. We will use a synergistic, multidisciplinary approach that bridges molecular physiology with structural biology. We will study SM tissues from normal and knock-out mice, with an experimental design that allows for the decoupling of the Ca2+-dependent phenomena from RhoA dependent regulation. Using X-ray crystallography, NMR, SAXs and DXMS, we will dissect the molecular mechanism by which the multidomain GEFs and GAPs are regulated in vitro and in vivo. Our research will explain fundamental aspects that control SM contractility and this knowledge may be used to design novel therapies for widespread diseases such as hypertension and asthma.
Diseases like hypertension, coronary and cerebral vasospasm, which compromise blood flow to the heart and brain respectively, as well as asthma which is caused by constriction of the airways, are all caused by abnormal contraction and relaxation of smooth muscle in these tissues. We are studying the role of specific proteins in the regulation of smooth muscle's response to increases in intracellular calcium, which is the primary stimulus for contraction. The results of our research may translate into novel treatments for these diseases.
|Lakshmikanthan, Sribalaji; Zieba, Bartosz J; Ge, Zhi-Dong et al. (2014) Rap1b in smooth muscle and endothelium is required for maintenance of vascular tone and normal blood pressure. Arterioscler Thromb Vasc Biol 34:1486-94|
|Derewenda, Urszula; Artamonov, Mykhaylo; Szukalska, Gabriela et al. (2013) Identification of quercitrin as an inhibitor of the p90 S6 ribosomal kinase (RSK): structure of its complex with the N-terminal domain of RSK2 at 1.8 A resolution. Acta Crystallogr D Biol Crystallogr 69:266-75|
|Utepbergenov, Darkhan; Derewenda, Zygmunt S (2013) The unusual mechanism of inhibition of the p90 ribosomal S6 kinase (RSK) by flavonol rhamnosides. Biochim Biophys Acta 1834:1285-91|
|Amin, Ehsan; Dubey, Badri Nath; Zhang, Si-Cai et al. (2013) Rho-kinase: regulation, (dys)function, and inhibition. Biol Chem 394:1399-410|
|Artamonov, Mykhaylo; Momotani, Ko; Utepbergenov, Darkhan et al. (2013) The p90 ribosomal S6 kinase (RSK) is a mediator of smooth muscle contractility. PLoS One 8:e58703|
|Khromov, Alexander S; Momotani, Ko; Jin, Li et al. (2012) Molecular mechanism of telokin-mediated disinhibition of myosin light chain phosphatase and cAMP/cGMP-induced relaxation of gastrointestinal smooth muscle. J Biol Chem 287:20975-85|
|Bielnicki, Jakub A; Shkumatov, Alexander V; Derewenda, Urszula et al. (2011) Insights into the molecular activation mechanism of the RhoA-specific guanine nucleotide exchange factor, PDZRhoGEF. J Biol Chem 286:35163-75|
|Zieba, Bartosz J; Artamonov, Mykhaylo V; Jin, Li et al. (2011) The cAMP-responsive Rap1 guanine nucleotide exchange factor, Epac, induces smooth muscle relaxation by down-regulation of RhoA activity. J Biol Chem 286:16681-92|
|Momotani, Ko; Artamonov, Mykhaylo V; Utepbergenov, Darkhan et al. (2011) p63RhoGEF couples Gýý(q/11)-mediated signaling to Ca2+ sensitization of vascular smooth muscle contractility. Circ Res 109:993-1002|
|Cierpicki, Tomasz; Bielnicki, Jakub; Zheng, Meiying et al. (2009) The solution structure and dynamics of the DH-PH module of PDZRhoGEF in isolation and in complex with nucleotide-free RhoA. Protein Sci 18:2067-79|